Kinetoplastid protozoa are the causative agents of devastating diseases in tropical and subtropical countries and include visceral and cutaneous leishmaniasis, sleeping sickness and Chagas disease. These protozoa are characterized by the presence of a novel form of mitochondrial DNA termed kinetoplast DNA or kDNA. This DNA consists of 5,000 to 10,000 minicircles catenated into a single DNA network along with 20 to 30 maxicircles. This proposal focuses on two areas: the identification of essential genes and proteins required for kDNA replication and the determination of features of minicircle and maxicircle replication mechanisms that relate to regulation of their replication during the cell cycle. Different aspects of this research will utilize the organism most appropriate for the question under investigation. One approach to identifying new genes involved in kDNA replication will exploit newly developed genetic tools for Trypanosoma brucei together with information from the T. brucei genome project. Alternate approaches based on protein-protein interactions will utilize Crithidia fasciculata and Leishmania tarentolae to take advantage of cloned genes, recombinant proteins and specific antisera against several replication proteins from these organisms and the sequence information from the Leishmania genome project. The second major aim of this proposal addresses the regulation of minicircle and maxicircle replication mechanisms in C. fasciculata. These studies take advantage of our experience with synchronized cultures of this trypanosomatid and the extensive prior studies on its kDNA replication mechanisms. Examination of the biochemical structure of maxicircle replication intermediates and the nucleotide sequence around the maxicircle replication origin will be important for determining which features of the replication mechanism are common to both minicircles and maxicircles. [unreadable] [unreadable]